1. Field of the Invention
The present invention relates to methods for producing taxane-type diterpenes including taxol which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like.
2. Description of the Prior Art
Taxol useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer, etc. is a taxane-type diterpene isolated and identified from Pacific yew (Taxus brevifolia NUTT), a plant belonging to the genus Taxus, the family Taxaceae. This compound has a complex ester group related to its activity. Taxol can be found in any part of the plant body of Pacific yew, and it has been reported that taxol content is the highest in the bark. Currently, taxol is collected from natural or cultured trees. However, Taxus plants are slow-growing plants taking more than 10 years to grow to a height of 20 cm above the ground. Further, peeling the bark results in the death of trees. Therefore, it is difficult to obtain a large quantity of taxol. If it is possible to synthesize taxol and taxane-type diterpenes such as baccatin III (a precursor of taxol) utilizing cell culture, that will be very advantageous for obtaining large quantities of these compounds easily without harvesting trees.
As prior art for taxol production, a semi-synthetic method to produce taxol from baccatin III or 10-deacetylbaccatin III, a precursor in the biosynthesis of taxol, is disclosed in the U.S. Pat. No. 5,015,744 issued to Holton et al. In Europe and the U.S., taxol derived from this semi-synthesis has been approved and used clinically. By using a method of plant cell culture, it is possible to produce raw materials for semi-synthesis, such as baccatin III, which can be utilized for taxol production by the above-described semi-synthetic process.
As a method of taxol production using cultured plant cells, a method has been patented in the U.S. (U.S. Pat. No. 5,019,504) in which taxol is produced from cultured cells of Pacific yew (Taxus brevifolia NUTT). According to this method, however, the yield of taxol is 1-3 mg/l which is insufficient for industrial production. Besides, the taxol productivity by cell culture is unstable. Although a primary cell of high productivity can be obtained by selection, it is difficult to maintain the taxol content of the cell through subculture [E. R. M. Wickremesine et al., World Congress on Cell and Tissue Culture (1992)].
Under circumstances, various methods have been tried to improve the productivity of taxol. For example, a method in which cultured cells of Taxus chinensis that has a high taxol content are used (U.S. Pat. No. 5,407,816); a method of using a continuous culture process (Japanese Unexamined Patent Publication No. 7-255495); and a method in which methyl jasmonate, an information transmission substance, is added to the medium (Japanese Unexamined Patent Publication No. 8-33490, EP 683232) may be enumerated. However, none of the above-described methods has been put into practical use. Further improvement of the productivity is desired.
Cultured cells of plants will not form single cells but produce cell clusters several ten micrometers to several millimeters in size even when cultured in a liquid medium under agitation because, generally, the cell walls adhering individual cells are firm and solid. In arbor plants such as Taxus plants used in the present invention, intercellular adhesion is particularly strong as a result of the development of secondary cell walls such as lignin.
Recent researches concerning the production of secondary metabolites in cultured plant cells have shown that the secondary metabolites vary depending on the size of these cell clusters [Y. Yamada and Y. Fujita, Handbook of Plant Cell Culture, vol. 1, edited by D. A. Evans et al., Macmillan Publishing Co., New York, pp. 717-728 (1983); Y. Hara et al., Planta Med., 55, pp. 151-154 (1989)]. However, no report has been made on the relation between the particle size of those cell clusters and the productivity of taxane-type diterpenes.
On the other hand, U.S. Pat. No. 5,344,775 discloses the following processes as a method for obtaining small cell clusters of a Taxus plant consisting of 1-10 cells:
(i) providing fragments of callus tissue from Taxus explants containing meristematic tissue physically supported on a support culture medium containing xcex1-napthaleneacetic acid as an auxin, and 6-benzylamino purine as a cytokinin;
(ii) culturing the callus tissue in a liquid medium containing the auxin and the cytokinin to produce a suspension of a plurality of clusters of 1-10 cells having limited intercellular adhesion;
(iii) plating cells removed from the cell suspension onto a surface of a support culture medium containing the auxin and the cytokinin; and
(iv) growing the plated cells from step (iii) on the support culture medium form pseudocallus cells, the pseudocallus cells being a loose, amorphous aggregation of cells lacking differentiated vascular or organ tissues and lacking clearly defined meristematic zones, the cell aggregation having poor intercellular adhesion, extreme friability, and falling apart into numerous individual cells and small cell clusters when mechanically disturbed, the pseudocallus cells showing an initial rate of mass doubling in cell growth medium that is greater than the mass doubling of callus tissue of step (i), and exhibiting the property of producing higher levels of taxanes than that produced by callus tissue of step (i).
However, it is extremely difficult to obtain small cell aggregations which consist of 1-10 cells and yet which can grow or produce secondary metabolites. (Cultured cells of Taxus plants are usually 20-30 xcexcm in diameter and, thus, the diameter of a cell aggregation consisting of 10 cells is estimated to be less than 100 xcexcm.) It is impossible to obtain such aggregations unless the medium and culture conditions are elaborately combined as disclosed in the above-mentioned U.S. patent.
In cultured cells of a number of Taxus plants including those cells used by the present inventors, small clusters of 1-10 healthy cells cannot be obtained by merely intensifying the agitation conditions employed in liquid culture. What can be obtained by fractionation using a sieve 100 xcexcm in opening is the debris of aged cells alone which do not have the ability to grow or to produce secondary metabolites.
Therefore, the major point of the separation of small cell clusters as disclosed in the above U.S. patent resides in the preparation of such callus tissue that liberates small cell clusters easily. Thus, this patent is different in basic thought and process from the present invention which defines conditions of shaking and agitation to liberate small cell clusters. In other words, according to the specification of the above U.S. patent, the shaking and agitation conditions of the patent to liberate small clusters consisting of 1-10 healthy cells fall within the range of conventional methods. Unlike the conditions of the present invention, the conditions employed in that U.S. patent are not beyond the range of conditions used in conventional cell culture.
Furthermore, considering actual, industrial culture processes, it is expected in any of the above-described methods to increase the productivity of finally obtained taxane-type diterpenes by employing the so-called two-step culture in which preculture and main culture are performed under different conditions. Preculture is conducted for the purpose of growing cells, and main culture is conducted for the purpose of producing taxane-type diterpenes of interest such as taxol. In the production of taxane-type diterpenes by conventional cell culture methods, improvement of conditions in main culture has been emphasized as seen in the above-mentioned 3 patents relating to the improvement of taxol productivity. For example, an elicitor or information transmission substance is added; or culture process is improved. With respect to preculture, no specific conditions preferable for the improvement of the final productivity of a compound of interest are known.
As roles of preculture, the first one is to increase the growth rate of cells so that cells are supplied to main culture as much as possible. At the same time, preculture is required to increase the latent ability of cells to produce taxane-type diterpenes so that the cells, when transferred, will produce diterpenes efficiently in main culture. However, the importance of preculture conditions from the viewpoint of increasing the cells"" latent ability has not been recognized at all as described above.
Generally, as parameters to control or regulate oxygen supply to plant cells or the like in suspension culture, mass transfer volume coefficient (kLa) and dissolved oxygen concentration (DO) can be enumerated, for example. Shaking rate and gas feeding rate cannot be used universally since they are influenced by the shape of the culture vessel and the amount of medium used, though they are easily determined and advantageous in that sense. Thus, they are totally meaningless parameters in terms of cultural engineering. It has been known that the productivity of a product of interest (amount of accumulation/cell) is influenced by kLa and DO in full-scale culture (Y. Fujita and Y. Hara, Agric. Biol. Chem., 49, pp. 2071-2075 (1985)]. However, the influence of these parameters in preculture has not been examined to date.
It is an object of the invention to improve the productivity of taxane-type diterpenes in methods for producing the same by culturing plant cells.
As a result of intensive and extensive researches toward the solution of the above assignment, the present inventors have found that (i) in culturing taxane-type diterpene-producing plant cells, the use of small cell clusters increases the productivity of taxane-type diterpenes; and (ii) preculturing of taxane-type diterpene-producing plant cells under enriched oxygen supply increases the latent ability of the resultant cells to produce taxane-type diterpenes, improving in turn the productivity of the diterpenes in main culture. Thus, the present invention has been achieved.
The present invention relates to a method for producing a taxane-type diterpene(s) by culturing cells of a taxane-type diterpene-producing plant, comprising one or both of the the following steps (a) and (b) to increase the ratio of those cell clusters suitable in size for diterpene production:
(a) at least one operation for removing large cell clusters is conducted with a sieve and/or a filter during preculture of the cells to be supplied to main culture for the production of a taxane-type diterpene(s) or at the time of cell transfer from one preculture to the subsequent preculture;
(b) the cells are cultured under strong agitation conditions.
The cell cluster suitable in size for diterpene production has a mean diameter ranging from 0.12 mm to 1.6 mm, preferably from 0.12 mm to 1.0 mm, the mean diameter being represented by an intermediate value between the major axis and the minor axis of the cell cluster.
Preferably, the ratio of the cell clusters suitable in size for diterpene production is increased to 65% or more, especially 80% or more, relative to the gross fresh weight of the cells.
The present invention also relates to a method for producing a taxane-type diterpene(s) by culturing cells of a taxane-type diterpene-producing plant, comprising:
preculturing the cells in a liquid medium under oxygen supply conditions wherein the mass transfer volume coefficient (kLa) of oxygen against the liquid medium is 10 hxe2x88x921 or more;
supplying the resultant cells to main culture for the production of a taxane-type diterpene(s); and
recovering the taxane-type diterpene(s) from the resultant culture.
Further, the present invention relates to a method for producing a taxane-type diterpene(s) by culturing cells of a taxane-type diterpene-producing plant, comprising:
preculturing the cells in a liquid medium under conditions wherein the dissolved oxygen concentration (DO) of the liquid medium is retained at 30% or more of the saturated oxygen concentration at the temperature employed in the culture;
supplying the resultant cells to full-scale culture to produce a taxane-type diterpene(s); and
recovering the taxane-type diterpene(s) from the resultant culture.